AMP Assay

ABSTRACT

The invention provides a method for detecting AMP, which method comprises treating a sample with adenylate kinase, nucleoside-diphosphate kinase, and a phosphate donor under conditions such that when AMP is present in the sample ATP is formed; and detecting the ATP so formed, preferably with the light-producing luciferase-luciferin reaction.

This invention relates to the enzymatic detection of the presence oramount of AMP (adenosine 5′-monophosphate), and other analytes relatedto the production or consumption of AMP.

In an AMP assay described by Brovko et al. (1994) AnalyticalBiochemistry, 220, 410-414, AMP is converted to ADP with CTP as aphosphate donor in a reaction catalyzed by adenylate kinase.Subsequently, ADP is converted to ATP with phosphoenolpyruvate as aphosphate donor in a reaction catalyzed by pyruvate kinase. This enzymeis known to be inhibited by phosphate and has an absolute requirementfor monovalent cations such as potassium or ammonium ions. The ATPformed thereby is detected with the light-producing luciferase-luciferinreaction. In the presence of ATP and O₂, luciferase catalyzes theoxidation of luciferin, producing light that can be quantitated with aluminometer. Additional products of the reaction are AMP, pyrophosphate,and oxyluciferin. For improved sensitivity, the three enzymes of theassay are co-immobilized onto cyanogen bromide-activated agarose.Nevertheless, the reported detection limit for this assay is as high as15 pmol.

In an AMP assay described in U.S. Pat. No. 5,891,659, AMP is convertedto ATP with phosphoenolpyruvate and pyrophosphate as phosphate donors ina reaction catalyzed by pyruvate phosphate dikinase. This reaction iscoupled to the light-producing luciferase-luciferin reaction. However,the two reactions are incompatible in that pyrophosphate is an inhibitorof luciferase activity. Furthermore, the pH optimal for the activity ofpyruvate phosphate dikinase is suboptimal for luciferase activity.Therefore, a high concentration of luciferase is required, preferably atleast 500 μg/ml, which makes the assay expensive. Pyruvate phosphatedikinase has an absolute requirement for monovalent cations such aspotassium or ammonium ions.

In U.S. Pat. No. 6,335,162, AMP is detected by converting it to ATP withphosphoribosylpyrophosphate as a pyrophosphate donor in a reactioncatalyzed by phosphoribosylpyrophosphate synthetase. The ATP produced isdetected with the light-producing luciferase-luciferin reaction.However, the phosphoribosylpyrophosphate synthetase reaction proceeds tothe direction of ATP synthesis only under narrowly defined reactionconditions not compatible with the subsequent luciferase reaction.

Therefore, it is an object of the present invention to provide an assayfor AMP, which does not suffer from the disadvantages of the knownprocedures which make their use difficult or expensive.

Thus, the invention in one embodiment provides a method for detectingAMP, which method comprises treating a sample with adenylate kinase,nucleoside-diphosphate kinase, and a phosphate donor under conditionssuch that when AMP is present in the sample ATP is formed; anddetetecting the ATP so formed, preferably with the light-producingluciferase-luciferin reaction.

In another embodiment, the invention provides a reagent for detectingAMP, which reagent comprises adenylate kinase, nucleoside-diphosphatekinase, and a phosphate donor, and which preferably further comprisesluciferase and luciferin.

In yet another embodiment, the invention provides a kit for detectingAMP, which kit comprises in a packaged combination adenylate kinase,nucleoside-diphosphate kinase, and a phosphate donor, and whichoptionally further comprises luciferase and luciferin.

Appropriate conditions for the method of the present invention includeappropriate component concentrations, solution temperature, ionicstrength, and incubation time. Such conditions also include the presenceof any appropriate additional substances, such as enzyme cofactors,stabilizers, and buffering agents. Appropriate incubation conditions fora given enzyme, or coupled enzyme system, are generally known in the artor are readily determined using standard methods known in the art.

In preferred embodiments the phosphate donor is a nucleosidetriphosphate or an analogue thereof, and in particularly preferredembodiments, the phosphate donor is dCTP (2′-deoxycytidine5′-triphosphate) available for example from Amersham Biosciences(Piscataway, N.J.).

In some embodiments, the ATP formed is detected with a detection systemother than the luciferase-luciferin reaction. In a commerciallyavailable ATP kit (#366-A, Sigma, St. Louis, Mo.), a combination of twoenzymes, phosphoglycerate kinase and glyceraldehyde phosphatedehydrogenase, are used to catalyze the formation of NAD from NADH inthe presence of the ATP formed, which is detected as a decrease inNADH-dependent UV absorbance (340 nm) or fluorescence emission (460 nm),as described further in U.S. Pat. No. 6,335,162, incorporated herein byreference. Another detection system for the ATP formed is based on theuse of a FAD synthetase-active system in conjunction with FMN togenerate FAD. This detection system is fully described in U.S. Pat. No.4,806,415, incorporated herein by reference.

In preferred embodiments, the ATP formed is detected by theluciferase-luciferin reaction. In the presence of ATP and O₂, luciferasecatalyzes the oxidation of luciferin, producing light. Additionalproducts of the reaction are AMP, pyrophosphate and oxyluciferin. Thelight can be detected by a luminometer or similar light-sensitiveinstrument, or more specifically, by a photomultiplier, a photodiode, acharged coupled device (CCD), or the like.

Preferred luciferase is recombinant firefly luciferase available forexample from Promega (Madison, Wis.) and, as a kit component, fromMolecular Probes (Eugene, Oreg.).

By detecting is meant detecting the presence or amount.

By adenylate kinase is meant the enzyme of EC 2.7.4.3, or any enzyme,ribozyme, or the like, capable of catalyzing the conversion of AMP toADP with a nucleoside triphosphate as a phosphate donor. In the presentinvention, the preferred adenylate kinase is that of EC 2.7.4.3, and inparticular, myokinase from chicken muscle available from Sigma (St.Louis, Mo.). In an application that requires incubation at an elevatedtemperature, it may be more preferable to use a thermostable adenylatekinase such as myokinase from Bacillus stearothermophilus also availablefrom Sigma.

By nucleoside-diphosphate kinase is meant the enzyme of EC 2.7.4.6, orany enzyme, ribozyme, or the like, capable of catalyzing the conversionof ADP to ATP with a nucleoside triphosphate as a phosphate donor. Inthe present invention, the preferred nucleoside-diphosphate kinase isthat of EC 2.7.4.6, and in particular, Saccharomycescerevisiaenucleoside-diphosphate kinase available from Sigma. In anapplication that requires incubation at an elevated temperature, it maybe more preferable to use a thermostable nucleoside-diphosphate kinasesuch as that from Pyrococcus furiosus, described in the published U.S.Patent Application 20010031470, incorporated herein by reference.

In addition to AMP, this invention also relates to the enzymaticdetection of other analytes related to the production or consumption ofAMP.

For example, RNA in a sample is detected by adding to the sample aribonuclease that hydrolyzes RNA to nucleoside monophosphates includingAMP that is then detected by the method of the present invention. On theother hand, a ribonuclease in a sample is detected by its ability tohydrolyze added RNA to nucleoside monophosphates including AMP that isthen detected by the method of the present invention.

The method for detecting AMP of the present invention may be based onthe following reactions, wherein dCTP as a phosphate donor may bereplaced by another nucleoside triphosphate or an analogue thereby:

AMP+dCTP→ADP+dCDP, catalyzed by adenylate kinase;

ADP+dCTP→ATP+dCDP, catalyzed by nucleoside-diphosphate kinase; and

ATP+O₂+luciferin AMP+pyrophosphate+CO₂+oxyluciferin+light, catalyzed byluciferase.

A preferred phosphate donor such as dCTP is substrate for both adenylatekinase and nucleoside-diphosphate kinase, but may not be substrate forluciferase.

The three reactions shown above are preferably coupled together, suchthat the ATP consumed in the light-producing luciferase-luciferinreaction is regenerated in the reactions catalyzed by adenylate kinaseand nucleoside-diphosphate kinase.

Thereafter, these three reactions occur simultaneously, continuously andrepeatedly.

It is confirmed that under appropriate conditions, the intensity oflight produced by the above coupled reaction system is substantiallyconstant for at least 30 minutes.

However, in the above coupled reaction system, pyrophosphate willaccumulate in the course of time, eventually leading to the inhibitionof light production.

Therefore, appropriate conditions for the method of the presentinvention may include an agent to break down any pyrophosphategenerated. Preferably said agent is inorganic pyrophosphatase (EC3.6.1.1) such as that from Saccharomyces cerevisiaeavailable from Sigma(St. Louis, Mo.).

The following examples are intended to illustrate the present inventionand in no way limit any aspect of the invention.

EXAMPLE 1

Reaction Buffer:

25 mM aqueous Tricine buffer, pH 7.8, 5 mM MgSO₄, 0.1 mM EDTA, and

0.1 mM sodium azide (Molecular Probes, Component E of A-22066, Lot64B1-1).

Reagent A:

200 μM dCTP (sodium salt, Amersham Biosciences, 272062, Lot 8620),

20 units/ml myokinase (Sigma, M5520, Lot 012K7485), and

20 units/ml nucleoside-diphosphate kinase (Sigma, NO₃₇₉, Lot 119H7455)in Reaction Buffer.

Reagent B:

5 mM D-luciferin (sodium salt, Molecular Probes, Component A of A-22066,Lot 64B1-1), 50 μg/ml luciferase, firefly recombinant (Molecular Probes,Component B of A-22066, Lot 64B1-1), and

10 mM dithiothreitol (DTT) (Molecular Probes, Component C of A-22066,Lot 64B1-1) in Reaction Buffer.

Reagent C:

45 volumes of Reagent A, and

10 volumes of Reagent B.

EXAMPLE 2

A sample containing 10 pmol AMP (sodium salt, Sigma, A1752, Lot042K7000) in 45 μl of Reaction Buffer was mixed in a polystyrene testtube (Sarstedt, 55476) with 45 μl of Reagent A in which 200 μM dCTP wasreplaced with either 500 μM AMPCPP (α, β-methyleneadenosine5′-triphosphate lithium salt, Sigma, M6517, Lot 101K7028), 500 μM dGTP,500 μM dCTP or 500 μM dTTP (sodium salts, Roche, Mannheim, Germany,1969064, Lot 90704020). After 30 min of incubation at room temperature,10 μl of Reagent B was added, and after 15 min, the tube was read in aBerthold 9509 luminometer for 10 s. RLU=relative light units.Appropriate controls (no AMP, no phosphate donor) were included. Thereadings for duplicate reactions (I and II) are shown in Table 1. TABLE1 AMP (pmol) Phosphate donor RLU I RLU II Average RLU — — 163 166 164 10— 172 169 170 — AMPCPP 374 368 371 10 AMPCPP 18362 19805 19084 — dGTP2140 1957 2048 10 dGTP 54745 61576 58160 — dCTP 690 698 694 10 dCTP59787 56687 58237 — dTTP 9278 10565 9922 10 dTTP 65304 60376 62840Table 1 shows that a nucleoside triphosphate or an analogue thereof canserve as a phosphate donor for detecting AMP in the present invention.Of the phosphate donors tested, dCTP has the highest signal-to-noiseratio.

EXAMPLE 3

A sample containing 0.1-10 pmol AMP (sodium salt, Sigma, A1752, Lot042K7000) or 10 pmol ATP (disodium salt, Molecular Probes, Component Dof A-22066, Lot 64B1-1) in 45 μl of Reaction Buffer was mixed in apolystyrene test tube (Sarstedt, 55476) with 45 μl of Reagent A. After30 min of incubation at room temperature, 10 μl of Reagent B was added,and after 15 s, 15 min and 30 min, the tube was read in a Berthold 9509luminometer for 10 s. RLU=relative light units. No-analyte control(blank) was included. The average readings for duplicate reactions areshown in Table 2. TABLE 2 Average RLU AMP (pmol) 15 s 15 min 30 min —726 666 646 0.1 1360 1287 1280 0.5 3684 3543 3309 1   6938 6367 6192 5  31993 27954 26668 10   63780 53392 51614 ATP (10 pmol) 63199 57107 51788Table 2 shows that Reagent A efficiently converts AMP present in asample to ATP, which is subsequently detected with Reagent B. The lightsignal is directly proportional to the quantity of AMP and substantiallyconstant for at least 30 minutes. With the method of the invention, fmolquantities of AMP can be detected.

EXAMPLE 4

A sample containing 0.1-10 pmol AMP (sodium salt, Sigma, A1 752, Lot042K7000) or 10 pmol ATP (disodium salt, Molecular Probes, Component Dof A-22066, Lot 64B1-1) in 45 μl of Reaction Buffer was mixed in apolystyrene test tube (Sarstedt, 55476) with 55 μl of Reagent C, andafter 10, 20 and 30 min at room temperature, the tube was read in aBerthold 9509 luminometer for 10 s. RLU=relative light units. No-analytecontrol (blank) was included. The average readings for duplicatereactions are shown in Table 3. TABLE 3 Average RLU AMP (pmol) 10 min 20min 30 min — 512 510 500 0.1 900 890 878 0.5 2460 2274 2004 1   46164396 3772 5   17802 17814 16730 10   34874 33200 32031 ATP (10 pmol)35456 33659 31268Table 3 shows that AMP in a sample can be detected in a single step withReagent C. The light signal is directly proportional to the quantity ofAMP and substantially constant for at least 30 minutes.

EXAMPLE 5

A sample containing 0.1-10 pmol AMP (sodium salt, Sigma, A 1752, Lot042K7000) or 10 pmol ATP (disodium salt, Molecular Probes, Component Dof A-22066, Lot 64B1-1) in 45 μl of Reaction Buffer was mixed in apolystyrene test tube (Sarstedt, 55476) with 55 μl of Reagent C, andafter 1, 2, 3, 4 and 5 min at room temperature, the tube was read in aBerthold 9509 luminometer for 10 s. RLU=relative light units. No-analytecontrol (blank) was included. The average readings for duplicatereactions are shown in Table 4. TABLE 4 Average RLU AMP (pmol) 1 min 2min 3 min 4 min 5 min — 508 504 488 486 489 0.1 866 860 871 848 860 13844 4184 4182 4144 4103 10 34336 37216 37166 37118 36792 ATP (10 pmol)37374 35982 35849 35014 34700Table 4 shows that AMP in a sample can be detected in a single stepalmost immediately after adding Reagent C. The light signal is directlyproportional to the quantity of AMP.

1. A method for detecting AMP, characterized in that it comprises (a)treating a sample with adenylate kinase, nucleoside-diphosphate kinase,and a phosphate donor under conditions such that when AMP is present inthe sample, ATP is formed; and (b) detetecting the ATP formed.
 2. Amethod according to claim 1, wherein the ATP formed is detected with thelight-producing luciferase-luciferin reaction.
 3. A method according toclaim 1 or 2, wherein said phosphate donor is a nucleoside triphosphateor an analogue thereof.
 4. A method according to claim 1 or 2 whereinsaid phosphate donor is dCTP.
 5. A reagent for detecting AMP,characterized in that it comprises adenylate kinase,nucleoside-diphosphate kinase, and a phosphate donor.
 6. A reagentaccording to claim 5 further comprising luciferase and luciferin.
 7. Areagent according to claim 5 or 6, wherein said phosphate donor is anucleoside triphosphate or an analogue thereof.
 8. A reagent accordingto claim 5 or 6 wherein said phosphate donor is dCTP.
 9. A kit fordetecting AMP, characterized in that it comprises in a packagedcombination adenylate kinase, nucleoside-diphosphate kinase, and aphosphate donor.
 10. A kit according to claim 9 further comprisingluciferase and luciferin.
 11. A kit according to claim 9 or 10, whereinsaid phosphate donor is a nucleoside triphosphate or an analogue thereof12. A kit according to claim 9 or 10, wherein said phosphate donor isdCTP.